Journal of Microbiology

Research Article

Efficiency of reverse genetics methods for rescuing severe acute respiratory syndrome coronavirus 2: Chang-Joo Park, Taehun Kim, Seung-Min Yoo, Myung-Shin Lee, Nam-Hyuk Cho, Changhoon Park; J. Microbiol. 2025;63(2):e2411023. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2411023

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Abstract PDF: Bacteria-free reverse genetics techniques are crucial for the efficient generation of recombinant viruses, bypassing the need for labor-intensive bacterial cloning. These methods are particularly relevant for studying the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This study compared the efficiency of three bacteria-free approaches—circular polymerase extension reaction (CPER) with and without nick sealing and infectious sub-genomic amplicons (ISA)—to bacterial artificial chromosome (BAC)-based technology for rescuing SARS-CoV-2. Significant differences in viral titers following transfection were observed between methods. CPER with nick sealing generated virus titers comparable to those of the BAC-based method and 10 times higher than those of the standard CPER. In contrast, ISA demonstrated extremely low efficiency, as cytopathic effects were detected only after two passages. All rescued viruses exhibited replication kinetics consistent with those of the original strain, with no significant deviation in replication capacity. Furthermore, the utility of CPER and ISA in genetically modifying SARS-CoV-2 was demonstrated by successfully inserting the gene encoding green fluorescent protein into the genome. Overall, this study underscores the potential of bacteria-free methods, such as CPER and ISA, in advancing SARS-CoV-2 research while highlighting their significant differences in efficiency.

Research Support, Non-U.S. Gov'ts

Mutational inactivation of OprD in carbapenem-resistant Pseudomonas aeruginosa isolates from Korean hospitals: Chi Hyun Kim , Hee Young Kang , Bo Ra Kim , Hyejin Jeon , Yoo Chul Lee , Sang Hwa Lee , Je Chul Lee; J. Microbiol. 2016;54(1):44-49. Published online January 5, 2016; DOI: https://doi.org/10.1007/s12275-016-5562-5

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This study investigated the mechanisms underlying the carbapenem resistance of bloodstream isolates of Pseudomonas aeruginosa obtained from two Korean hospitals. Of the 79 P. aeruginosa isolates, 22 and 21 were resistant to imipenem and meropenem, respectively. The 22 imipenem-resistant P. aeruginosa isolates were classified into 7 sequence types (STs) and 13 pulsotypes. Twelve imipenem-resistant isolates from one hospital were found to belong to the international clone ST111. Two imipenem-resistant P. aeruginosa ST235 isolates carried the blaIMP-6 gene, but the remaining 20 isolates did not produce carbapenemases. Mutations in the oprD gene and a related decrease in gene expression were found in 21 and 5 isolates, respectively. However, all imipenemresistant P. aeruginosa isolates showed no significant expression of OprD in the outer membrane as compared with that of carbapenem-susceptible PAO1 strain. Overexpression of genes associated with efflux pumps, including mexB, mexD, mexF, and mexY, was not found in any imipenem-resistant isolate. One imipenem-resistant P. aeruginosa isolate overexpressed the ampC gene. Our results show that the low permeability of drugs due to the mutational inactivation of OprD is primarily responsible for carbapenem resistance in bloodstream isolates of P. aeruginosa from Korean hospitals.

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Development of a Chimeric Strain of Porcine Reproductive and Respiratory Syndrome Virus with an Infectious Clone and a Korean Dominant Field Strain: Jung-Ah Lee , Nak-Hyung Lee , Sang-Won Lee , Seung-Yong Park , Chang-Seon Song , In-Soo Choi , Joong-Bok Lee; J. Microbiol. 2014;52(4):345-349. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4074-4

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Abstract

The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 106 TCID50/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.

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Porcine Reproductive and Respiratory Syndrome Virus Engineered by Serine Substitution on the 44th Amino Acid of GP5 Resulted in a Potential Vaccine Candidate with the Ability to Produce High Levels of Neutralizing Antibody
Jong-Chul Choi, Min-Sik Kim, Hwi-Yeon Choi, Yeong-Lim Kang, In-Yeong Choi, Sung-Won Jung, Ji-Yun Jeong, Min-Chul Kim, Andrew Y. Cho, Ji-Ho Lee, Dong-Hun Lee, Sang-Won Lee, Seung-Yong Park, Chang-Seon Song, In-Soo Choi, Joong-Bok Lee
Veterinary Sciences.2023; 10(3): 191. CrossRef
Porcine Reproductive and Respiratory Syndrome Virus: Immune Escape and Application of Reverse Genetics in Attenuated Live Vaccine Development
Honglei Wang, Yangyang Xu, Wenhai Feng
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Evaluation of the Cross-Protective Efficacy of a Chimeric PRRSV Vaccine against Two Genetically Diverse PRRSV2 Field Strains in a Reproductive Model
Chang-Gi Jeong, Amina Khatun, Salik Nazki, Seung-Chai Kim, Yun-Hee Noh, Sang-Chul Kang, Dong-Uk Lee, Myeon-Sik Yang, Nadeem Shabir, In-Joong Yoon, Bumseok Kim, Won-Il Kim
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NOTE] Evaluation of a Fosmid-Clone-Based Microarray for Comparative Analysis of Swine Fecal Metagenomes: Soo-Je Park , Dong-Hwan Kim , Man-Young Jung , So-Jeong Kim , Hongik Kim , Yang-Hoon Kim , Jong-Chan Chae , Sung-Keun Rhee; J. Microbiol. 2012;50(4):684-688. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2115-4

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Abstract: Glass slide arrayed with fosmid clone DNAs generated from swine feces as probes were fabricated and used as a metagenome microarray (MGA). MGA appeared to be specific to their corresponding target genomic fragments. The detection limit was 10 ng of genomic DNA (ca. 106 bacterial cells) in the presence of 1000 ng of background DNA. Linear relationships between the signal intensity and the target DNA (20–100 ng) were observed (r2=0.98). Application of MGA to the comparison of swine fecal metagenomes suggested that the microbial community composition of swine intestine could be dependent on the health state of swine.

Diversity of Bacterial Community in Freshwater of Woopo Wetland: Keun Sik Baik , Seong Chan Park , Eun Mi Kim , Kyung Sook Bae , Jae-Hyung Ahn , Jong-Ok Ka , Jongsik Chun , Chi Nam Seong; J. Microbiol. 2008;46(6):647-655. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0135-x

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Abstract: Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.

Generation of Infectious Transcripts from Korean Strain and Mild Mottle Strain of Potato Virus X: Sun Hee Choi , Ki Hyun Ryu; J. Microbiol. 2008;46(5):502-507. Published online October 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0078-2

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Abstract: Full-length cDNAs of two different strains of Potato virus X (PVX-Kr and PVX-Mo) have been directly amplified by long template reverse transcription polymerase chain reaction (RT-PCR) using the 5’-end primer containing a SP6 or T7 RNA promoter sequence and the virus-specific 3’-end primer, and then constructed in plasmid vectors. Capped in vitro transcripts from cloned full-length cDNAs as well as those RTPCR amplicons proved to be infectious systemically on tobacco plants. Symptom expression on tobacco plants from PVX-Mo transcripts was faster and severer than that from PVX-Kr. In replication stability test of transcripts derived from PVX clones, progeny viruses showed stable replication according to sequencing through passages. This highly infectious transcript system from the full-length cDNA clones for PVX can be useful for recombinant molecules for functional analysis of viral proteins in plant-virus interaction study as well as for expression of foreign protein in planta.

Phenotypic and Genotypic Differences of the Vancomycin-Resistant Enterococcus faecium Isolates from Humans and Poultry in Korea: Jae Young Oh , Seunghun An , Jong Sook Jin , You Chul Lee , Dong Teak Cho , Je Chul Lee; J. Microbiol. 2007;45(5):466-472.; DOI: https://doi.org/2588 [pii]

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Abstract: A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates (58 isolates from patients and 40 isolates from poultry) were compared based on their antimicrobial susceptibility, Tn1546 element organization, and pulsed-field gel electrophoresis (PFGE) patterns. This comparison aided in determining the relationships between the groups of isolates. All the VREF isolates harbored the vanA gene; however, 29 (29.6%) of the isolates exhibited the VanB phenotype-vanA genotype. Furthermore, the VREF isolates from humans and poultry exhibited distinct antimicrobial resistance patterns. The PCR mapping of the Tn1546 elements exhibited 12 different transposon types (A to L). The VREF isolates of poultry were classified into types A to D, whereas the human isolates were classified into types E to L. A PFGE analysis demonstrated a high degree of clonal heterogeneity in both groups of isolates; however, the distinct VREF clones appeared in each group of isolates. The deletion of the vanX-vanY genes or insertion of IS1216V in the intergenic region from the vanX-vanY genes is directly associated with the incongruence of the VanB phenotype-vanA genotype in human VREF isolates. These data suggest that the VREF isolates exhibit distinct phenotypic and genotypic traits according to their origins, which suggests that no evidence exists to substantiate the clonal spread or transfer of vancomycin resistance determinants between humans and poultry.

Molecular Characterization of Pseudomonas aeruginosa Isolates Resistant to All Antimicrobial Agents, but Susceptible to Colistin, in Daegu, Korea: Yoo Chul Lee , Byung Jun Ahn , Jong Sook Jin , Jung Uk Kim , Sang Hwa Lee , Do Young Song , Won Kil Lee , Je Chul Lee; J. Microbiol. 2007;45(4):358-363.; DOI: https://doi.org/2560 [pii]

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Abstract: Multi-drug resistant Pseudomonas aeruginosa has been implicated in a variety of serious therapeutic problems in clinical environments. Among the 968 P. aeruginosa isolates obtained from two hospitals in Daegu, Korea, we acquired 17 isolates that were resistant to all available tested antimicrobial agents, with the exception of colistin (colistin-only sensitive). We characterized the antimicrobial susceptibilities, metallo-β-lactamases, and epidemiological relatedness among the colistin-only sensitive P. aeruginosa isolates. All colistin-only sensitive isolates were positive in the modified Hodge test and imipenem-EDTA synergy test, thereby indicating the production of metallo-β-lactamases. 11 isolates from the secondary hospital and six isolates from the tertiary teaching hospital harbored blaVIM-2 and blaIMP-1, respectively. The pulsed-field gel electrophoretic analysis of the SpeI-digested DNA from P. aeruginosa isolates indicated that two different clones of colistin-only sensitive P. aeruginosa originated from each hospital, and had spread within the hospital environment. Overall, colistin-only sensitive P. aeruginosa was detected in Korea for the first time, but no pan-drug resistant bacteria were identified. Nationwide surveillance is required in order to monitor the emergence of colistin-only sensitive or pan-drug resistant bacteria.

Diversity of Microorganisms in Decaying Maize Stalks Revealed by a Molecular Method: Ming-Xia Yang , Han-Bo Zhang; J. Microbiol. 2007;45(4):367-370.; DOI: https://doi.org/2558 [pii]

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Abstract: Microbial diversity in decaying maize stalk was characterized by constructing and analyzing rRNA gene clone library. Total 47 OTUs were obtained from 82 bacterial clones, including Proteobacteria (64.6%), Actinobacteria (30.5%), Bacteroidetes (2.4%) and Firmicutes (2.4%). Most proteobacterial clones were members of Rhizobium, Pseudomonas and Stenotrophomonas. Eighty-four percent of Actinobacteria was related to Microbacterium. Only 14 OTUs were identified from 124 fungal clones, including Ascomycota (88%) and Basidiomycota (12%). Sixty percent of Ascomycota were members of Eupenicillium and Paecilomyces but all Basidiomycota were close to Kurtzmanomyces nectairei.

Comparison of Bacterial Composition between Human Saliva and Dental Unit Water System: Eun-Hyoung Jeon , Ji-Hye Han , Tae-Young Ahn; J. Microbiol. 2007;45(1):1-5.; DOI: https://doi.org/2500 [pii]

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Abstract: The bacterial compositions between the dental unit water system and human saliva were characterized and compared by direct sequence analysis of 16S rDNA clone libraries. Based on the species richness estimation, bacterial diversity in the dental unit water system (DUW) was more diverse than that of the human saliva (HS). The Chao1 estimates of species richness in HS and DUW samples were 12.0 and 72.4, respectively. The total numbers of OTUs observed in the combined libraries accounted for 83% (HS) and 59% (DUW) of the Chao1 diversity estimate as defined at the 80% similarity threshold. Based on the sequence analysis, the phylum Proteobacteria was the major group in both clone libraries at phylum level. DUW clone library contained 80.0% Proteobacteria, 8.0% Bacteroides, 4.0% Nitrospira, 4.0% Firmicutes, 2.0% Planctomycetes and 2.0% Acidobacteria. On the other hand, human saliva (HS) clone library contained 55.5% Proteobacteria, 36.1% Firmicutes and 8.4% Bacteroides. The majority of bacteria identified belonged to phylum Proteobacteria in both samples. In dental unit water system (DUW), Alphaproteobacteria was detected as the major group. There was no evidence of the bacterial contamination due to a dental treatment. Most sequences were related to microorganisms derived from biofilm in oligotrophic environments.

Bacterial Diversity in the Human Saliva from Different Ages: Jung-Gyu Kang , Seong Hwan Kim , Tae-Young Ahn; J. Microbiol. 2006;44(5):572-576.; DOI: https://doi.org/2438 [pii]

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Abstract: To obtain primary idea on oral bacterium species that are generally present in periodotally healthy Koreans, the oral bacterial flora in the saliva of four periodontally healthy Koreans at different ages (5, 32, 35, 65) was investigated in this study. For this investigation, 16SrRNA gene clone libraries were generated from the saliva of the four healthy Koreans, and 50 clones were randomly selected from each saliva clone library and sequenced. Totally, 37 different kinds of bacterial 16S rRNA gene sequences were identified based on sequence homology search through GenBank database. The 37 kinds of saliva clone sequences were classified to 14 genera and 2 uncultured and 1 unidentified bacteria. Among the 14 identified genera, Streptococcus, Prevotella, and Veillonella were common genera, and Streptococcus was dominant genus that accounted for 7 different species. Among the seven Streptococcus species, S. salivarius appeared as the most common species. More numbers of species belonging to the genera Streptococcus and Prevotella was present in saliva from ages 32 and 35. While saliva from ages 5 and 65 showed more numbers of species belonging to the genera Rothia, including potential pathogenic species. Overall, saliva of a young child and a senior showed higher bacterial diversity than that of young adults.

Cloning and sequence determination of α-tubulin, β-tubline and Flagellar Calmodulin cDNAs of Naegleria gruberi: Choi, Youn Jeong , Park, Hye Lee , Lee, Joo Hun; J. Microbiol. 1995;33(1):40-45.

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Abstract: Five cDNAs encoding two α-tubulins(α13 and α15), two β-tubulins(β5 and β5), and one flagellar calmodulin (Cal-1) were cloned from naegleria gruberi NB-1 and their nt sequences were determined. The α13(EMBL number X81049) and β1(EMBL number X81050) contained a complete open reading frame for α-tubulin and β-tubulin, respectively. The other three clones (α15, β5 and Cal-1) had a part of coding region and a 3’ untranslated region of the respective genes. The α13, β1 and Cal-1 had no homologous sequences in the coding regions and in the 3’ untranslated sequences. However, the α13 and β1 shared an eight nucleotide (AATACAAA) sequence in front of the respective initiation codons. The AATACAAA sequence was also found in N. gruberi strain NEG α-tubulin cDNA clone(αpT1) at the same position. Comparison of the α13 to the αpT1 revealed another stretch of identical sequence, which is 30 nts long, in the 5’ untranlated region.

Quantitative Analysis of Expressed Genes in Aspergillus Oryzae by Sequencing 3'-directed cDNA Clones: Hwang, Hyun Ah , Lee, Dong Whan , Kim, Jong Hwa , Lee, Tae Kyoo , Yang, Moon Sik , Chae, Keon Sang; J. Microbiol. 1998;36(2):111-117.

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Abstract: Sequence analysis of randomly selected 3'-directed cKNA clones has been known to be one of the most powerful methods of examining the genes highly expressed in a tissue or cell type. We constructed a 3'-directed cDNA libraty from Aspergillus oryzae mycelia, and sequenced 345 randomly selected 3'-directed cDNA clones. Determined nucleotide sequences, not shorter than 30nt, were compared with one other to generate gene signatures (GSs) and were then compared with GenBank entries to analyze sequence similarity to known genes. A GS for the most highly expressed gene appeared six times, one GS five times, five GSs four times, five GSs three times and 22 GSs twice. In total, 324 clones yielded 268 GSs consisting of 34 redundant GSs appeaning at least twice and 234 solitary ones. Forty-three GSs showed similarities ranging from 60% to 99% with known sequences from Genbank. A considerable number of A. oryzae GSs mateched those obtained from the sexual structures of A. nidulans suggests that A. oryzae may not be phylogentically distant from A. nidulans and that A. oryzae may have a sexual life cycle from the ancient period.

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